ABSTRACT Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. There is an increased risk of CHIKV pandemic due to climate and ecological change and international travel. Currently, there is no approved vaccine. Previous experimental approaches include the US Army-developed IND vaccine, 181/25, that contains two attenuating mutations in the E2 protein. The 181/25 vaccine showed high immunogenicity in Phase II clinical trial; however, adverse reactions justify the need for safety improvement. Medigen has developed a DNA-launched, live-attenuated vaccine, termed iDNA, in which the full-length genome of the 181/25 IND vaccine virus is transcribed from a CMV promoter on a plasmid. Injection of the plasmid intramuscularly launches the live-attenuated vaccine virus. This approach has the advantages of DNA vaccines, including the encoding of the vaccine virus by a genetically stable DNA plasmid, and the high efficacy of live-attenuated vaccines. To address the need for improved safety of the 181/25 vaccine, we propose a genomic rearrangement as an additional attenuating mutation. This genomic rearrangement (RA181/25) does not change immunogenic epitopes but is resistant to reversion since multiple, independent mutations would be needed for reversion. In Specific Aim 1, we prepare the rearranged (RA181/25) CHIKV iDNA vaccine and evaluate it in vitro for plaque phenotype, growth curve, and genetic stability by next generation sequencing (NGS), as compared to non- rearranged iDNA and the 181/25 IND vaccine virus. In Specific Aim 2, RA181/25 CHIKV iDNA is evaluated in two mouse models (inbred C57BL/6 and outbred CD-1) as compared to the non-rearranged CHIKV iDNA and the 181/25 IND vaccine virus. We propose to use electroporation, microneedles, and liposomal formulations to optimize the iDNA vaccination, and we will evaluate safety, immunogenicity (neutralizing antibody and cell-mediated responses), and protective efficacy against CHIKV challenge in both animal models. The successful proof-of-concept demonstration of a safety advantage of the RA181/25 CHIKV iDNA vaccine would advance a novel single-dose vaccine with many advantages for protecting populations at risk for CHIKV infection, as well as for rapidly controlling CHIKV outbreaks.